OBJECTIVETo evaluate the effects of Icariin (ICA) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in vitro and in vivo.

METHODSAn enzymatic digestion block was used in vitro to culture hPDLSCs, which were separated and purified by limited dilution cloning. The hPDLSCs were identified using cell-surface markers and cocultured with 1 x 10(-7) mol.L-1 ICA solution. The proliferation ability of these cells was determined by thiazolyl blue tetrazolium bromide (MTT) assay. After staining with alkaline phosphatase (ALP), osteogenesis was detected by enzyme-linked immunosorbent assay. Osteoblast-related genes were analyzed by reverse transcription-polymerase chain reaction. Alizarin red staining was performed to measure the level of calcium deposition. The hPDLSCs were cocultured with 1 x 10(-7) mol.L-1 ICA and nano-hydroxyapatite scaffolds in vivo before transplantation into subcutaneous tissues of nude mice. Osteogenic abilities were histochemically analyzed after 30 days of induction.

RESULTSThe hPDLSCs were affected by 1 x 10(-7) mol.L-1 ICA, and MTT assay showed that the proliferation of the groups treated with ICA in vitro was better than that of the control groups on the second day. The ALP activity of the treated hPDLSCs was significantly enhanced after cell culture for 3, 5, and 7 days. The gene expression of osteoblastic markers was also significantly enhanced after 7 days. The deposition of mineralization after incubation with 1 x 10(-7) mol.L-1 ICA increased compared with the control after cell culture for 14, 21, and 28 days. Furthermore, the bone expression of the treatment groups in vivo was significantly enhanced compared with that of the control groups.

CONCLUSIONTreatment with 1 x 10(-7) mol.L-1 ICA can significantly promote proliferation and differentiation of hPDLSCs in vitro and in vivo. ICA can effectively function as a bioactive growth factor in periodontal tissue engineering to replace traditional growth factors.

Alkaline Phosphatase ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Coculture Techniques ; Enzyme-Linked Immunosorbent Assay ; Flavonoids ; Humans ; Mice ; Mice, Nude ; Osteoblasts ; Osteogenesis ; Periodontal Ligament ; Stem Cells